Anti-viral N-substituted pyrimidines

ABSTRACT

Nucleoside analogues having a ring-open structure, of general formula: ##STR1## where R and R&#39; may be hydrogen, silyl groups, substituted alkyl groups, benzyl groups and the like, and X is an optionally substituted base such as guanine or adenine, have been shown to exhibit anti-viral and other biological acivities at non-toxic levels.

This is a division of pending application Ser. No. 302,790, filed Sept.16, 1981, which is a CIP of U.S. Ser. No. 187,631 for Sept, 16, 1980,now U.S. Pat. No. 4,347,360, incorporated herein by reference.

BACKGROUND OF THE INVENTION

This invention relates to novel compositions and chemical compounds andprocesses for their preparation. More particularly, it relates to novelring-open nucleoside and nucleotide analogues and the like, which showbioregulation activity, e.g. antiviral activity and processes for theirsynthesis.

BRIEF REFERENCE TO THE PRIOR ART

Nucleosides comprise a D-ribose or 2-deoxy-D-ribose sugar unit,chemically bonded to a purine or pyrimidine base selected from adenine,cytosine, guanine, thymine and uracil, via a nuclear nitrogen atom ofthe base. Since they are units of nucleic acids found naturally inliving cells, it has been speculated previously that nucleosides andnucleotides and their related analogs might have potential aschemotherapeutic agents. Any practical value they may have, however, isoften greatly reduced by their ready deamination in vivo by deaminases.Stuides have been conducted to determine the relationship betweenstructure and activity for both substrates and inhibitors of adenosinedeaminase, some such studies involving ring-opened analogues ofnucleosides. To date, however, despite several promising reports ofnovel compounds, no such compounds have been produced and developed forchemotherapeutic use, with the possible exception of a-cycloguanosine,described in U.S. Pat. No. 4,146,715 Schaeffer.

SUMMARY OF THE INVENTION

The present invention relates to antivirally active dinucleotide andnucleoside analogues, processes for their preparation, andpharmaceutically acceptable compositions thereof for administration tomammals to treat viral infections. The nucleoside analogues of thepresent invention are N-substituted purine and pyrimidine compoundscorresponding to the general formula: ##STR2## wherein X represents auracil group, a 5-fluorouracil group, a cytosine group, a 5-azacytosinegroup, an adenine group, a guanine group or a 2-N-acetylguanine group;and R and R' are independently selected from hydrogen, benzyl andtert.butyldimethylsilyl, with the proviso that, when X represents anadenine group, at least one of R and R' represents other than hydrogen.

It will be appreciated that the compounds according to the presentinvention are closely analogous in structure and groupings to naturallyoccurring nucleosides and nucleotides. The essential chain arrangementsand lengths are maintained. The appropriate O and OH functional groups,which in biological environments actively bind to biological centers,are maintained in their natural sequences and disposition relative tothe base, but optionally modified with "protecting" groups. Indeed, thegroups adjacent to the bases are so similar in chemical constitution todeoxyribose compounds that they can assume the essential conformation ofthe deoxyribose ring under appropriate conditions. The fundamentaldifference is that the compounds of the present invention lack thestructural rigidity of carbohydrate ring, which renders them uppredictably different in properties and behaviour. Also, the C-4'position is not chiral, in compounds of formula I, so that stereiosomersdo not arise. Each hydroxyl is primary. There can be no syn-antiisomerism about the glycosidic bond.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The preferred bases X in the compounds of the present invention are thepurine bases adenine and guanine. Compounds of adenine abound in nature,and show wide ranges of biological activity. It is among adenine andquanine compounds of the present invention that the most biologicallyactive compounds are found. Test procedures for characterization andevaluation, e.g. with specific enzymes, are well established inconnection with adenine compounds.

In contrast with natural adenosine compounds and most of the previouslyreported synthetic analogues thereof, the adenine compounds of thepresent invention are resistant to attack by adenosine deaminase enzymesfound in most mammalian tissue, and deactivation thereby. Natural andpreviously reported synthetic analogues of adenosine compounds areattacked by this enzyme in vivo, with the result that the amine group atC6 on the prime ring is hydrolyzed to hydroxyl, forming thecorresponding inosine compound, the majority of which are biologicallyinactive. With compounds subject to this reaction, the results of testsof biological activity carried out in vitro do not provide any usefulguide to in vivo activity.

The compounds of the present invention, however, are very poorsubstrates for adenosine deaminase, and consequently do not deaminate invivo, at least to any sigificant extent. Consequently, test resultsobtained in vitro are also obtainable in vivo.

Most preferred of compounds of formula I is that in which R and R' areboth hydrogen and X is adenine or guanine, namely the compound ofgeneral fomula: ##STR3##9-[[2-hydroxy-1-(hydroxymethyl)-ethoxy]-methyl]adenine, and thecorresponding compounds in which one or both of R and R' representdimethyl-tert.-butylsilyl, or of formula ##STR4##

Compounds of general formula I may be made by coupling the appropriatelyhalogenated base with the appropriate alkyl residue. The synthesis maybe initiated by treating 1,3-dichloro-2-propanol with sodium benzylateunder a nitrogen atmosphere followed by trioxymethylene and HCL toprepare the chloromethoxy derivative, of 1,3-dibenzyloxy-2-propanol carebeing taken to remove excess water. This derivative may be coupled tothe appropriately halogenated base, such as 6-chloropurine, in DMF usingtriethylamine as acid scavenger. Treatment of the chloro compound soformed with methanolic ammonia in a steel reaction bomb gives the6-amino derivative. The product may be debenzylated to give a compoundof formula I, e.g. with hydrogen over palladium oxide in methanol.Protecting groups, if desired, are put on by standard, known methods.Alaternatively, halogenated alkyl residues may be coupled withhalogenated or non-halogenated purine or pyrimidine base compounds.

Several of the compounds of the present invention show anti-viralactivity, accompanied by low cell toxicity, rendering them potentiallyuseful in therapeutic compositions to combat specific viral invaders ofliving mammalian cells. For example, the compound9-[[2-hydroxyl-1-(hydroxymethyl)-ethoxy]-methyl]adenine, is activeagainst herpes simplex virus, influenza or virus, and against vesicularstomatitis virus. The compound G*(9-[[2-hydroxy-1-(hydroxymethyl)ethoxy]methyl]-guanine is extremelyactive against herpes simplex virus, and in fact shows an activity ofmagnitude much greater than that of acycloguanosine, as well as beingactive also against a broader range of viruses than acycloguanosine.Also, mono-O-tert-butyldimethyl silyl-9-[[2-hydroxy-1-(hydrov-methyl)ethoxy]-methyl]-adenine is active against influenza virus. Inboth cases, these compounds are active to combat the viruses, andprevent or at least substantially inhibit the replication thereof, at adosage level at which they are non-toxic to mammalian cells.

Certain compounds of the aforementioned formula I in which the purine orpyrimidine base group X is substituted on the nucleus are also ofinterest as potential pharmacological agents e.g. anti-virals. Specificsuch compounds are those in which X represents uracil substituted at the5-position with fluoro or hydroxymethyl; guanine or adenine substitutedat the 8-position with halogen (especially but not limited to bromine),thio or amino, 5-fluorouracil, 5-aza-cytosine, 2-N-acetyl guanine, etc.

It will of course be understood that the present invention extends tocover pharmaceutically acceptable salts of the compounds describedherein.

Compounds according to the present invention may be administered to apatient parenterally, interthecally applied topically as ointment,cream, aerosol or powder, or even on occasion given as eye or nose dropsor orally. In general, methods of administration and dosage formulationsthereof follow the known, published methods used with known antiviraldrugs such as acycloguanosine, Ara-A and Ivdr. Effective unit doses foradministration of the compositions interthecally or parenterally,calculated as free base, are suitably in the range from about 0.1-100 mgper k mammal body weight, most suitably in the 0.5-20 mg per kg mammalbody weight, and most preferably about 5 mg per kg, on the basis of adosage administered from 2-4 times per day.

Orally administrable compositions are preferably in fine powder orgranule form, with diluting and/or dispersing and/or surface activeagents, e.g. in water or in a syrup dispersion, or as tablets orcapsules. Solutions of the compounds in distilled water or saline, e.g.isotonic solutions and optionally buffered with phosphate, ofconcentration 1-20% preferably 2-15% and most preferably around 10%, aresuitable for parenteral or interthecal administration. Ointments(topical or cream) may be compounded for treatment of externalinfections, e.g. with an oil in water cream base, in a concentration offrom about 0.1-10% preferably up to about 3%, most preferably about 1%w/v a tive ingredient. They may be compounded with paraffin oil, withpetrolatum to form emulsion optionally with a surfactant for stabilizingpurposes, or with dimethyl-sulfoxide.

The invention is further illustrated in the following non-limitativeexamples.

EXAMPLE 1 Preparation of9-[[2-hydro-1-(hydroxymethyl)-ethoxy]methyl]adenine(III)

6.5 m.moles of 6-chloropurine was condensed with1,3-dibenzyloxy-2-chloromethoxypropane (6.5 m.moles) indimethylformamide (4 ml) containing triethylamine (6.5 m.moles) at 25°C. for 16 hours. The product is formed,1,3-dibenzyloxy-2-(6-chloropurine)-methoxy propane, was isolated fromTLC plates as an oil, and subsequently heated in a steel bomb at 90° C.for 20 hours with 60 ml. methanol saturated (0° C.) with ammonia. Thesolvents were evaporated and 1,3-dibenzyloxy-2-adeninemethoxypropaneobtained on precipitation from ethanol with ether. The compound wasdebenzylated using palladium oxide inmethanol at 25 psi of hydrogen for20 hours. The catalyst was removed by filtration, and on concentratingand cooling the methanol solution, the product9-[[2-hydroxy-1-(hydroxymethyl)ethoxy]-methyl]-adenine (III)crystallized as a white solid. The overall yield from 6-chloropurine was27% melting point 184°-186° C.

m/e (molecular weight) 239, λ_(max) ^(EtOH) =259 nm

R_(f) 0.12 (CHCl₃ -Et OH, 4:1).

EXAMPLE 2

From compound III prepared according to Example 1,mono-O-tert.butyldimethylsilyl-9-[[2-hydroxyl-1-(hydroxymethyl)ethoxy]methyl]-adenine (compoundX) and bis-O-tert.butyldimethylsilyl-9-[[2-hydroxy-1-(hydroxymethyl)ethoxy]methyl-adenine (compound XI)were prepared by reaction with appropriate controlled amounts oftert.butyl silyldimethyl chloride, in the standard manner for protectionhydroxyl groups, according to methods of nucleoside synthesis, followedbystandard procedures for separating the two products. ##STR5##whereTBDMS represents tert.butyldimethylsilyl.

EXAMPLE 3

Compound III prepared according to Example 1 was tested for activityagainst viruses. The tests were conducted in the normal way growingmammalian cells in an appropriate medium on a culture disc. In thecontrols, viral cells were sprinkled onto the growing cell cultures andsubsequent growth thereof observed. In the test experiments, both viralcells and compounds according to the invention were sprinkled onto thegrowing cell cultures.

The viral plaque growth was observed. Reduction in the numbers and sizeof the plagues growing on the cells indicates that the added compound ispreventing the reproduction of the viruses.

Compound III was found to be active to inhibit reproduction of herpessimplex virus. At a dosage of 300 micrograms of compound per ml ofmedium, the plague area was reduced by 70%, without showing any evidenceof toxicity towards the mammalian cells growing in the culture.

Compound III was also found to be active to prevent reproduction of theVSV(vesicular stomatitis virus), reducing the plaque numbers by 70% at adosage of 1 mg per ml. Again, no evidence of toxicity to mammaliancells, at this same dosage level, was detected.

EXAMPLE 4

Compound X, prepared according to Example 2, was tested by the proceduredescribed in Example 3 for activity against influenza A virus. It wastested at dosage levels of 0.1, 1.1 and 11 psg per ml, and at each ofthese levels was active to inhibit reproduction of the viral cellswithoutdemonstrating toxicity towards the mammalian cells. At higherdosage levels(110 micrograms per ml) it was toxic to the mammaliancells.

When compound X was similarly tested against the herpes simplex virus,it demonstrated toxicity towards mammalian cells at dosage level of 300microg s/ml, without indicating selective activity against the virus.

Compound XI, in similar tests, indicated a very high level of toxicitytowards the mammalian cells, at a dosage level of 30 micrograms per ml.

EXAMPLE 5 Preparation of1-[[2-benzyloxy-1-(benzyloxymethyl)ethoxy]methyl]-5-fluorouracil

5-Fluorouracil (1.0 g, 0.0077 mole) and some crystals of ammoniumsulfate were suspended in 1,1,1,3,3,3-hexamethyldisilazane (HMDS) (15 g)and brought to reflux with stirring. After 50 minutes the base haddissolved and the excess HMDS was evaporated under reduced pressure toyield syrup 15 of silyl-protected base (structure not determined). Thesyryp was dissolved in 1,2-dichloroethane (80 ml) and anh drous stannicchloride (0.4 ml) was added. 1,3-Dibenzyloxy-2-chloromethoxy propane(0.007 m mole)from a stock solution was added and the solution wasstoppered and allowed to stand at room temperature overnight. Thereaction mixture was shaken with aqueous sodium bicarbonate and thephases were separated. The aqueousphase was extracted with chloroform.The combined organic phases were washed once with water, dried withanhydrous sodium sulfate and evaporatedunder reduced pressure to yield4.57 g of material. The nmr spectrum of thecrude material suggested thatthe proportion of desired compound in the mixture was 88%. The materialwas mixed with 15 g of silica (Fisher-S-662 ) and applied to a silicacolumn (93×2.0 cm). The column was eluted with 1% methanol in chloroform(250 ml), 3% methanol in chloroform (200 ml), and 5% methanol inchloroform (700 ml). When colored material began to appear fractionswere collected and the first 16 test tubes contained the desiredmaterial. The fractions were individually evaporated to syrupswhich onstanding overnight crystallized. Some methanol was added, the crystalswere broken up and the solvent was removed with a pasteur pipet. Thecrystals were washed once with methanol to yield 0.886 g of crystals and2.231 g of mother liquid residues. The residues were crystallizedfromcarbon tetrachloride and the resultant mother liquor residues wereapplied to preparative tlc plates and eluted with 5% methanol inchloroform. Eventually 1.81 g (0.0044 mole, 62%) of crystalline1-[[2-benzyloxy-1-(benzyloxymethyl)ethoxy]methyl]-5-fluorouracil wasobtained. An analytical sample was obtained by recrystallizing the abovematerial (tlc still showed an impurity) from a minimum of hot ethanol.Thecrystals gave: mp. 84°-86° C. and UV (EtOH) spectrum with _(max) 265nm.

The resultant compound, hereinafter referred to as 5F-benzyl-U*, has thestructural formula: ##STR6##

EXAMPLE 6 Preparation of5-Fluoro-1-[[2-hydroxy-1-(hydroxymethyl)ethoxy]methyl]-uracil

5F-benzyl-U* prepared as described in Example 6, (0.656 g, 0.00158 mole)was dissolved in ethanol (26 ml). Fresh palladium black (10 ml) wasadded,followed by cyclohexane (13 ml). After half-hour, tlc showed thatmost of the starting material was gone but there was still a fair amountof monobenzyl compound present. After 5 hours tlc showed that thereaction was complete and the mixture was filtered and the Pd black waswashed withethanol. The solution was evaporated under reduced pressureto yield 0.413 g of syrup which crystallized after the addition of somemethanol. The sample was dissolved in 3 ml of hot ethanol and then thevolume was reduced to 1 ml by blowing with nitrogen. The resultantcrystals (contained in a Greg tube) were washed 3 times with ethanol andthe residual solvent was removed by centrifugation to yield 0.185 g(0.00079 mole, 50%) of crystals. The mother liquor yielded another 0.089g (0.00038mole, 24%) of crystals. A second recrystallization gave mp.126°-128° C. and a UV spctrum (EtOH) _(max) 266 nm. The nmr spectrum inDC₃ OD and TMS gave: 3.58 (m, 5H, --CH₂ CH CH₂ --), 5.28 (s, 2H, OCH₂N.sub..), 7.85 (d, 1H, J_(F6) =6.0 Hz, H-6).

The resultant compound, hereinafter referred to as 5FU*, has thestructuralformula: ##STR7##

EXAMPLE 7 Preparation of5-az-1-[[1-hydroxymethyl-[2-hydroxy]ethoxy]methyl]cytosine

5-Azacytosine (5.0 g, 0.0446 mole) and some ammonium sulfate (100 mg)were suspended in HMDS (40 ml) and brought to reflux with stirring.After 20 minutes more ammonium sulfate was added and 20 minutes laterthe mixture became clear. The excess HMDS was evaporated under reducedpressure to yield a white solid, silyl-protected 5-azacytosine, whichwas used withoutfurther purification. The solid was dissolved in DCE(100 ml) and anhydrousstannic chloride (3.5 ml) was added. Then1,3-dibenzyloxy-2-chloromethoxy propane (0.040 mole) was added and thesolution was allowed to stand overnight at room temperature. Thereaction solution was poured into aqueous bicarbonate, diluted withchloroform and shaken. The resultant precipitate was removed byfiltration through celite. The phases were separated. The aqueous phasewas extracted once with chloroform. The organic phases were combined,washed once with water, dried with anhydroussodium sulfate andevaporated to yield 15 g of syrup. The syrup was dissolved in chloroform(20 ml) and applied to a tlc silica column (14.5×6.5 cm). The column wasfirst eluted with chloroform (450 ml).The solvent was changed to 5%methanol in chloforom and the collection of fractions (10-15 ml) wasbegun. The desired compound5-aza-1-[[2-benzyloxymethyl-[1-benzyloxy]-ethoxy]methyl]cytosine wasfoundin three groupings of fractions which were: 40-42 (1.48 g), 43-62(8.17 g) and 63-79 (0.42 g). The groupings were dissolved in 2.3 ml,12ml and 1.5 ml of hot ethanol respectively and seeded. The second sampleyielded 5.292g of crystals whereas the other two samples gave fewcrystals. Therefore the first and third sample were combined with themother liquor of the second sample and applied to a short silica column(4.0×6.3 cm) and first eluted with chloroform (125 ml) and then with 5 %methanol in chloroform when the collection of fractions (20 ml) wasbegun. The desiredmaterial appeared in fractions 7-8 (1.35 g) and 9-11(1.9 g). The 1.9 g sample was dissolved in ethanol (3 ml) and 0.917 g ofcrystals resulted. The yield of compound was 6.209 (0.0157, 39%). Thecrystals (6.209 g) wereaugmented by crystals (1.461 g) from anotherexperiment and recrystallized from ethanol (10 ml) to give 6.95 g ofwhite crystals. A sample was recrystallized twice from ethanol and itgave: mp 120°-122.5° C. The UV spectrum gave: _(max) (EtOH) 228, 236,(H₂ O) 241, (pH 1) 251, (pH 13) 250. The nmr spectrum (CDCl₃) gave: 3.52(d, 4H, J=5.5 Hz, CH₂ CHCH₂ --), 4.02 (m, 1H, --CH₂ CHCH₂ --), 4.45(s,4H, 2×PhCH₂ --), 5.30 (s, 2H, OCH₂ N), 5.95 (bs, 1H, HNH(, 7.27 (m, 11H,2×PhCH₂ --, HNH), 8.07 (s, 1H, H-6).

The above produced compound5-aza-1-[[2-benzyloxymethyl[1-benzyloxy]-ethoxy]methyl]cytosine (6.432g),0.0162 mole was dissolved in ethanol (200 ml, warmed). Used palladiumoxide(8 g) was added followed by cyclohexene (100 ml). The stirredmixture was brought to reflux and 3 hours later tlc suggested that thereaction was slow. Therefore fresh palladium oxide (2 g) was added andreflux was continued. After a total time of 22 hours some material hadprecipitated out but the tlc still showed some starting material and themonobenzyl analogue. The material was filtered and the residue waswashed with hot 95% ethanol until no more white precipitate was present.The filtrate and washings were combined and evaporated to yield about4.5 g of material. The material was recrystallized from hot ethanol andsome water (to help dissolve) to yield slightly greenish crystals (0.816g, 0.00377 mole, 23.3%) which gave mp 181°-5° C. and the filtrate wasgreen. The filtrate was evporated and the residue was dissolved in hotethanol (3.5 ml) and seeded. Crystals did not form. The material wasshaken with water and chloroform and the phases were separated. Theaqueous phase was co-evaporated with ethanol to yield 2 g of material.Crystallization in the usual way was not successful. The tlc showed anumber of components with the desired compound comprising about 30-40%of the mixture. The material was applied to 4 prep. tlc plates anddeveloped with 50% methanolin chloroform. The desired band was elutedwith 25% methanol in chloroform to yield 0.5 g of material.Crystallization in the usual way yielded only a trace of crystals. Thefirst crop of crystals were recrystallized from water (1 ml) and ethanol(7 ml) to give white crystals which gave: mp 189.5°-191° C. the desiredcompound gave UV spectrum: _(max) (EtOH) 220 nm, (H₂ O) 220, 245S,(pH 1) 250 (pH 13) 248. Thenmr spectrum (CD₃ OD+5 drops DMSO-d6+TMS)gave: 3.43-3.83 (m, 5H,--CH₂ CHCH₂ --), 5.37 (s, 2H, OCH₂ N), 8.27 (s,1H, H-6).

The resultant compound is5-aza-1-[[1-hydroxymethyl[2-hydroxy]-ethoxy]methyl]cytosine, hereafterreferred to as 5-aza-C*, has the structural formula: ##STR8##

EXAMPLE 8 Preparation of1-[[2-benzyloxy-1-(benzyloxymethyl)]ethoxymethyl]cytosine

Cytosine 4 (5.0 g, 0.045 mole) was suspended in1,1,1,3,3,3-hexamethyldisilazane (80 ml, 60 g HMDS) and several crystalsof ammonium sulfate were added (molded on: G. Ritzmann & W. Pfleiderer,Chem. Ber. 106, 1401 (1973)). The stirred mixture was protected frommoisture and refluxed until a clear solution was obtained. If a clearsolution was not obtained after one-half hour of reflux the addition ofmore ammonium sulfate gave a clear solution after another 10 minutes ofreflux. The clear hot solution was connected to a water aspirator andthe excess HMDS was carefully removed on a hot water bath to yield awhite solid which was used in the next step without purification.

The 2,4-bis-(trimethylsilyl)cytosine was dissolved in dry DCE (200 ml)and stannic chloride (3.4 ml, 29.1 moles anhydrous freshly distilled)was added. Then 40 g of stock chloride 3 solution (40 moles) was addedand theyellow solution was allowed to stand overnight at roomtemperature (modeledon B. U. Niedballa & H. Verbruggen, Angew. Chem.

The resultant product is1-[[2-benzyloxy-1-(benzyloxymethyl)]ethoxymethyl]cytosine, hereinafterreferred to as dibenzyl C*, of structural formula: ##STR9##

EXAMPLE 9 Preparation of2-N-Acetyl-9-[[2-benzyloxy-1-(benzyloxymethyl)ethoxy]methyl]-guanine

2-N-Acetylguanine (1.93 g, 10 mmoles) and ammonium sulfate (100 mg) weresuspended in HMDS (20 ml). The stirred mixture was refluxed for 3 hourswhen it became clear. The excess HMDS was removed under reduced pressureon a hot water bath to yield a white solid, silyl-protected2-N-acetylguanine, which was used without further purification. Thewhite solid was dissolved in DCE (50 ml) and1,3-dibenzyloxy-2-chloromethoxy propane (5 mmoles) was added followed byfreshly distilled anhydrous stannic chloride (1 ml). The solution wasallowed to stand overnight at room temperature. The solution was pouredinto a mixture of aqueous sodiumbicarbonate and chloroform and shaken.The mixture was filtered through celite to remove the precipitate. Thephases were separated and the aqeousphase was extracted once withchloroform.

The combined organic phases were washed with water, dried with anhydroussodium sulfate and evaporated under reduced pressure to yield 2.19 g ofmaterial. The reaction product was dissolved in chloroform (5 ml) andapplied to a tlc silica column (9×6.5 cm). The column was first elutedwith chloroform (60 ml) and then the solvent was changed to 2% methanolin chloroform. Three reasonably pure fractions were obtained fromtesttubes 15 (0.047 g), 19-20 (0.148 g) and 23-27 (0.43 g). Each of thesamples were crystallized with ethanol and gave 20 mg., 64 mg and 250 mgof material respectively. On the basis of U.V. spectra the 250 mgmaterialwas determined to be2-N-acetyl-9-[[2-benzyloxy-1-(benzyloxymethyl)ethoxy]meth l]guanine.

It was recrystallized from ethanol and gave m.p. 142°-4° C. The U.V.spectrum gave: max (EtOH)257,281 nm min 227,272, max (H₂ O) 259,278(shoulder), (pH1)262, (pH13)263.

The compound is hereinafter referred to as N-acetyl benzyl G*, and hasthe structural formula: ##STR10##

EXAMPLE 10 Preparation of9-[[2-hydroxy-1-(hydroxymethyl)ethoxy]methyl]guanine

N-acetyl benzyl G* prepared as described in Example 10 (1.288 g, 0.00270mole) was dissolved in pyridine (1.5 ml) andconcentrated ammoniumhydroxide (6 ml) was added. The flask was tightly supported andput in awater bath set at 55° C. After 15 hours, crystals had precipitated whichwere filtered and washed with ethanol. The crystals gave m.p. 170°-177°C., were recrystallized from ethanol (60 ml) to yield 0.863 g (0.00191mole, 70.7%) of9-[[2-benzyloxy-1-(benzyloxymethyl)ethoxy]methyl]guanine, m.p. 180°-182°C.

The above prepared compound (0.665 g, 0.00139 mole) was dissolved inrefluxing ethanol (40 ml). Palladium oxide (0.67 g) was added followedbycyclohexane (20 ml). Reflux was continued and after 2 hours tlc showedthatthere was still much starting compound present. Therefore morepalladium oxide (0.6 g, Aldrich Gold Label). After 5.5 hours thereaction still seemed to progress slowly therefore palladium black (0.5g--was stored several months under water and now dried by filtration andwashing with ethanol) was added. After seven hours more cyclohexene (15ml) was added. After 12 hours the reaction was not complete according totlc but after 22.5 hours tlc showed that the reaction was complete. Thereaction mixturewas filtered hot and the catalyst was washed with hot 95ethanol. Upon cooling crystals were deposited which were filtered andwashed with 95% ethanol. The yield of crystals was 127 mg which did notmelt up to 360° C. although they had become dark brown in colour. Thecatalyststill had product absorbed, and so it was washed with hot 75%ethanol. The washing was combined with the motor liquor from above andevaporated underreduced pressure. The residue was dissolved in a hotmixture of water (2.5 ml) and ethanol (2.5 ml) and then more ethanol(17.5 ml) was added with heating. The solution was allowed tocrystallize. The crystals (155 mg) were filtered and washed withethanol. The crystals did not melt up to 360° C. The mother liquoryielded about 60 mg of residue. Thereforethe yield of product was 282 mg((0.00110 mole, 79%). The UV spectrum gave: _(max) (EtOH), 254, 270shoulder, (H₂ O) 252, 269 (shoulder), (pH 1) 254, 272 (shoulder) (pH 13)262.

The product is 9-[[2-hydroxy-1-(hydroxymethyl)ethoxy]methyl]guanine,hereinafter referred to as G*, of structural formula ##STR11##

EXAMPLE 11 Testing and evaluation of compounds against herpes virus

Herpes simplex virus (HSV) strains were grown and titrated at 36° C.inhuman fetal fibroblasts derived from fetal tissues, and used for viruswork before the tenth passage. Cells were grown and maintained in basalmedium Eagle (BME; Auto-Pow, Flow Laboratories) supplemented with 0.112%sodium bicarbonate, mM 1-qlutanine, 2 mg neomycin per 100 ml and 5-20%calf serum. 5% BME, as described hereinafter, indicates mediumcontaining 5 ml of calf serum in a total volume of 100 ml.

The titer of the HSV strains is determined by a plaque titration method(Roizman & Roane, "Virology", 15, 75-79, 1961). Tissue culture dishesare added with cells and used for assays when approximately 75%monolayer. Volumes (0.2 ml) of logarithmic dilutions of the strain areinoculated onto each of two tissue culture dishes, and absorbed for 1hr. with intermittent shaking, the inoculum removed, and 2 ml of 5% BMEcontaining 0.5% human immune serum globulin added. After a 48 hourincubation period at 36° C. in a 5% CO₂ atmosphere, the overlay mediumis removed and the cell sheets stained with a 0.05% aqueous crystalviolet solution. The number of plaques is counted, the duplicatesaveraged, and the number of plaque-forming units calculated.

The compounds are tested for activity against the herpes simplex strainsusing a stock solution of each compound freshly prepared by dissolving1.2mg in BME. Appropriate dilution of each compound are made in 5% BMEcontaining 0.5% human immune serum globulin just before usage.

Tissue culture dishes (35 by 10 mm) with approximately 75% cellmonolayer are inoculated with approximately 50 plaque-forming units ofHSV per 0.2 ml, and the virus adsorbed for 1 hour, with intermittentshaking. After removal of the inoculum, 2 ml of 5% BME with 0.5% immuneglobulin and three-fold dilutions of the appropriate drug are added. Oneset of dishes receives no drug and is used as a control. After a 48-hourincubation period, at 36° C. in a 5% CO₂ atmosphere, the overlay mediumis removed, the cells stained as described above, and plaques counted.Thecounts of replicate plates are averaged, and the number of plaguesemergingin the presence of each drug dilution is calculated. Thereduction in plaque size caused by the concentration of the drug, ascompared with the relevant control, is also measured, visually. Areduction in plaque numberindicates that the added compound ispreventing the reproduction of the viral cells. A reduction in the areaof the growing plaque indicates inhibition of plaque growth, i.e.inhibition of viral reproduction, causedby the drug.

The results showed that G*, the compound of example II, wasoutstandingly effective against herpes simplex. It reduced plaque sizeby 25% at concentrations as low as 0.02 ugm/ml, by 50% at 0.1concentration and by 75% at 0.8 concentration. It reduced the plaquenumbers by 25% at 0.04 ugm/ml, by 50% at 0.1 concentration and by 75% at0.2 concentration. At the highest concentration level tested (250ugm/ml) it showed no evidence of toxicity towards the cells. When thecompound was used at concentrations of 2 ugm/ml and higher, no plaqueformation or growth was detected. Significant activity was detected atconcentrations as low as 0.007 ugm/ml.

The compound of example 8, 5-az-C*, also showed high activity againstthe HSV. It reduced plaque size by 25% at 5 ugm/ml, by 50% at 30 ugm/ml,and by 75% at 110 ugm/ml. It reduced plaque numbers by 25% at 8 ugm/ml,by 50 at 30 ugm/ml and by 75% at 140 ugm/ml. It showed no evidence oftoxicity at concentrations as high as 304 ugm/ml.

The compound of example 9, dibenzyl-C*,m also exhibited activity againstHSV, but to a lesser extent--25% plaque size reduction at 20 ugm/ml, 50%size reduction at 100 ugm/ml, 25% plaque number reduction at 20 ugm/mland50% plaque number reduction at 70 ugm/ml. The compound showedevidence of toxicity at concentrations of 305 ugm/ml.

The compound of example 6, 5F-benzyl-U*, showed activity against HSV,reducing plaque size by 25% at 10 ugm/ml, by 50% at 50 ugm/ml, andreducing plaque numbers by 25% at 70 ugm/ml, by 50% at 110 ugm/ml. Itshowed no evidence of toxicity up to concentrations of 110 ugm/ml.

The compound of example 10, N-acetyl-benzy G*, showed some activityagainstHSV, reducing plaque size by 25% at 60 ugm/ml, by 50% at 150ugm/ml, and reducing plaque numbers by 25% at 20 ugm/ml, by 50% at 130ugm/ml and by 75% at 310 ugm/ml.

Essentially similar results were obtained when compound G* was testedagainst 8 different strains of HSV type I, and 6 different strains ofHSV type II.

EXAMPLE 12 Further testing and evaluation of compounds

The compounds produced as described above were tested by standard plaquetitration methods to determine their activity against various othertypes of virus.

Tests for activity against the viruses were conducted as plaque tests,as described in Example 12 above. Compound G* showed activity againstVSV at low concentrations, and against coxsackie virus CVB3 at lowconcentrations.

Compound G* was also tested against the virus Varicella Zoster,responsiblefor chicken pox and shingles infections in man, by similarprocedures. It was found to be active against Varicella Zoster atconcentrations as low as 13 ugm/ml.

I claim:
 1. N-substituted pyrimidine compounds corresponding to theformula: ##STR12## wherein X represents a uracil group, a 5-fluorouracilgroup, or a cytosine group; and R and R' are independently selected fromhydrogen, benzyl and tert-butyldimethylsilyl.
 2. Compounds according toclaim 1, wherein X represents a pyrimidine moiety of formula ##STR13##3. A compound according to claim 2, which is1-[[2-benzyloxy-1-(benzyloxymethyl)ethoxy]methyl]-5-fluorouracil.
 4. Acompound according to claim 2, which is5-Fluoro-1-[[2-hydroxy-1-(hydroxymethyl)ethoxy]methyl]-uracil. 5.Compounds according to claim 1 wherein X represents a pyrimidine moietyof formula ##STR14##
 6. A compound according to claim 5 which is1-[[2-benzyloxy-1-(benzyloxymethyl)]ethoxymethyl]cytosine.
 7. Apharmaceutical composition suitable for administration to a mammal fortreatment of viral infections, selected from the group consisting ofherpes virus, influenza virus, and vesicular stomatitis virus,comprising as active ingredient 0.1 to 100 mg per kg of body weight, ofa compound according to claim 1.